A Study on Interactions between Different Viruses in the Pathogenesis of Cervical Cancer

Abstract

Background: With approximately 500,000 cases and over 270,000 deaths per annum, HPV-related invasive cervical cancer (ICC) is the second most common women’s cancer worldwide. In addition HIV and Adeno-Associated Virus Type 2 (AAV2) have also been shown to influence the progression of cervical dysplasia to ICC although their precise relationship to this process is unclear. This study aims to provide an increased understanding of how these three viruses, acting alone or in concert, may influence the pathogenesis of ICC. Materials and Methods: The prevalence of AAV2 DNA was examined by PCR in DNA extracted from 1013 liquid based cervical smears (LBCs) and 77 ICCs obtained from HIV positive and negative Kenyan women. Results were analysed with respect to cytology and socio-demographic factors. High fidelity PCR was also used to amplify 5 different HPV Type E2 genes from LBC DNAs, which were then cloned into a mammalian expression vector and transfected transiently in the cervical carcinoma cell lines HeLa, CaSki and C33A. E2 RNA expression levels, cell viability and TP53 protein levels were then measured. Results: There was no association of HIV and AAV2 but HPV and AAV2 were positively associated in women with normal or low grade dysplasia. AAV2 prevalence was markedly reduced in HPV positive women with either high-grade dysplasia or ICC although still present in ~14% of cases. PCR analysis of DNA from women with high grade dysplasia for the full length E2s of HPV types 52, 53, 58, 16 and 18 indicated that AAV2 was only detected in cases where full length E2s were also present. Ectopic expression of full length E2s from all these HPV types induced significant cell death in HeLa and CaSki but not in C33A cells. RT-PCR analysis of E2, E6, E7 and L1 mRNA in transfected HeLa and CaSki lines confirmed expression of all five E2 sequences and showed this was associated with downregulation of E6 and E7 and upregulated expression of L1. Immunofluorescence flow cytometry also showed increased expression of TP53. Conclusions: AAV2 prevalence in the cervix is not influenced by HIV but is positively associated with replication-competent episomal HPV. The lower prevalence of AAV2 in high grade dysplasia may be due to integration-associated reduced HPV replication as evidenced by the absence of full length E2. Alternatively AAV2 might also undergo integration into host DNA which might explain its persistence in 14% of ICCs. HIV is known to increase both the extent and diversity of infection with different HPV types and it is significant that E2s from these different types were shown to have promiscuous transcriptional regulatory activity in HPV16 and 18 expressing cell lines. This demonstrates the possibility that one episomal HPV type can regulate transcription of a second integrated HPV type when these are both present in a single cell. That a lower risk HPV such as type 53, which is more common in HIV positive women, can negatively regulate expression of viral oncogenes from higher risk HPVs such as 16 and 18, illustrates the potential for complex interactions between these viruses which may, in turn, regulate the pathogenesis of ICC.

Date of Award	1 Aug 2018
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Ian Hampson (Supervisor) & Lynne Hampson (Supervisor)